Multiple stimulus reversible hydrogels

ABSTRACT

A polymeric solution capable of gelling upon exposure to a critical minimum value of a plurality of environmental stimuli is disclosed. The polymeric solution may be an aqueous solution utilized in vivo and capable of having the gelation reversed if at least one of the stimuli fall below, or outside the range of, the critical minimum value. The aqueous polymeric solution can be used either in industrial or pharmaceutical environments. In the medical environment, the aqueous polymeric solution is provided with either a chemical or radioisotopic therapeutic agent for delivery to a specific body part. The primary advantage of the process is that exposure to one environmental stimuli alone will not cause gelation, thereby enabling the therapeutic agent to be conducted through the body for relatively long distances without gelation occurring.

This application is a continuation of U.S. patent application Ser. No.09/603,730 filed Jun. 23, 2000, now U.S. Pat. No. 6,660,247, issued Dec.9, 2003, which is incorporated herein by reference.

This invention was made with Government support under Contract No.DE-AC06-76RL01830 awarded by the U.S. Department of Energy. The U.S.Government has certain rights in the invention.

FIELD

The present invention relates in general to reversible gel compounds inwhich gelation is the result of response to a plurality of environmentalstimuli. More particularly, the present invention is directed topolymeric solutions which gel in response to exposure to a criticalminimum value of at least two environmental stimuli, such as in vivostimuli found in human or other mammalian bodies. The gelation responsecan be reversed by reducing the value of at least one of suchenvironmental stimuli to less than (or outside the range of) thecritical minimum value.

BACKGROUND

The use of reversible gelling compounds is well known in the art. In thecontext of this art, “gel” means a form of material between the liquidand solid state that consists of physically crosslinked networks of longpolymer molecules with liquid molecules trapped within the network—athree-dimensional network swollen by a solvent. If the solvent is water,the gel is termed a “hydrogel”.

Gels may be classified as either a chemical gel or a physical gel. Theformer are formed by chemical covalent bonds, resulting in a productcalled covalently cross-linked gels, and are not reversible. The latterare formed by secondary physical forces, such as hydrogen bonding orhydrophobic or charge interactions, and are reversible.

Commercially available block copolymers of poly(ethyleneoxide-b-propylene oxide-b-ethylene oxide) (PEO/PPO/PEO; Pluronics (BASF,Mount Olive, N.J.) or Poloxamers (ICI) are the best known examples ofreversible, thermally gelling polymers. PEO/PPO/PEO copolymers are afamily of more than 30 different nonionic surfactants, covering a widerange of liquids, solids and pastes with molecular weights ranging fromabout 1000 to 14,000. Concentrated solutions of PEO/PPO/PEO copolymersform reversible gels at high temperatures and revert to liquid stateupon lowering of temperature. Gelation temperature depends on polymercomposition and solution concentration.

Aqueous solutions of PEO/PPO/PEO copolymers demonstrate phasetransitions from sol to gel (low temperature sol-gel boundary) and gelto sol (high temperature gel-sol boundary) with monotonically increasingtemperature when the polymer concentration is above a minimum criticalvalue. The mechanism of gelation of PEO/PPO/PEO copolymers is stilluncertain.

Thermoreversible gels are also formed by several naturally occurringpolymers such as gelatin (a protein prepared from partial hydrolysis ofcollagen), polysaccharides such as agarose, amylopectin, carrageenans,Gellan™, and the like. All of this class of biopolymers form hydrogelswhen cooled. By contrast, cellulose derivatives gel by a differentmechanism: they are sols at low temperatures and become gels at hightemperatures. The sol-gel transition temperature is affected bysubstitutions at the hydroxyl group of cellulose.

Novel biodegradable triblock copolymers of polyethylene glycol andpoly(lactic/glycolic acid)(PEG/PLGA/PEG) were developed, and as aqueoussolutions exhibit sol to gel transition at body temperature. Anonresorbable thermoreversible gel based on copolymers ofN-isopropylacrylamide with acrylic acid (poly(NiPAAm-co-AAc)) have beendeveloped, and demonstrate reversible sol-to-gel transition atphysiological temperature ranges due to lower critical solutiontemperatures exhibited by polymers of the N-isopropylacrylamide.

As an example of a different gelling mechanism, charged, water solublepolymers may form reversible gels in response to pH change in solution.For example, chitosan solutions exhibit a sol-to-gel transition at a pHof about 7.0, when pH changes from slightly acidic to neutral. ThepH-triggered transition is slower than the transition caused by changesin temperature.

Chemically cross-linked gels are used extensively as matrices inchromatography and electrophoreses analytical methods to separatemolecules according to molecular weight and charge. Additionally,efforts have been made to deliver drugs to human patients via reversiblygelling polymers, as well as topical applications and for ophthalmicdelivery of therapeutic agents. It is known to use copolymer polyolswhich are available commercially under the trade name Pluronic™, asdescribed in U.S. Pat. No. 4,188,373.

In-situ gelling compounds have been proposed for use in implantation ofdrug delivery systems (for example, in cancer treatment), as well asinjectable matrices for tissue engineering. Stimulus induced in-vivogelation is a process that produces no toxic polymerization residues andresults in no heat generation.

For example, U.S. Pat. No. 5,252,318 discloses a reversibly gellingaqueous composition that undergoes significant viscosity changes tosimultaneous changes in both temperature and pH. The '318 composition iscomprised of a combination of at least two separate and distinctreversibly gelling polymers—one of which is temperature sensitive (suchas methyl cellulose or block copolymers of polyoxyethylene andpolyoxypropylene) and the other being pH sensitive (such as apolyacrylic acid). The composition is intended for use as dropinstillable, oral and injectable drug delivery vehicles, and fortopically applied lubricants, wetting agents and cleaning agents.

Other approaches to injectable polymers have included single-stimuluspolymers, as for example in U.S. Pat. No. 5,939,485. Gelation of theaqueous polymer solution is responsive to a change in a singleenvironmental stimulus, such as temperature, pH or ionic strength.

U.S. Pat. No. 4,732,930 discloses a chemically cross-linked gelcomposition comprised of a polymerized product that is obtained bypolymerization of isopropylacrylamide monomer, a source of metal ions, acrosslinking agent and a liquid medium. The product exhibits areversible phase transition function that results in a drastic volumechange in response to changes of the liquid medium compositiontemperature or ion concentration.

U.S. Pat. No. 5,525,334 discloses a method for vascular embolization byintroduction of an aqueous solution of a thermosensitive polymer whichgels in vivo at the body temperature of a patient. Obviously, such athermosensitive gelling response will be inoperative in a processwherein the polymer must travel a substantial distance within thepatient's body prior to gelation (such as when the gel is introducedthrough a catheter running from the femoral artery to the brain).

PCT published application number WO 99/56783 discloses a hydrogel forthe treatment of aneurysms, whereby the gel carries both a radiopaqueagent (permitting the radiogel to be visualized under fluoroscopy) and atherapeutic agent. The hydrogel is delivered through a catheter into ananeurysm, where the hydrogel becomes more viscous upon reaching bodytemperature or upon exposure to bodily fluids. The gelled compoundblocks flow into the aneurysm, and can be adapted to deliver a humangrowth factor to promote growth of a cellular layer across the neck ofthe aneurysm.

It is therefore an object of the present invention to provide a singleinjectable aqueous gelling solution that is sensitive to at least twoenvironmental stimuli, and more preferably, a compound that is sensitiveto at least two in vivo environmental stimuli in a human or othermammalian body. The compound of the present invention will gel whenexposed to critical minimum values of the environmental stimuli and ispreferably a reversibly gelling compound, such that when the criticalminimum values of all (or at a minimum, at least one) of theenvironmental stimuli fall below or outside the range of sensitivity,the gelled compound returns to an un-gelled condition.

The ideal multiple stimulus reversible hydrogel comprises anaqueous-based solution or compound having low viscosity at formationconditions, but exhibits rapid gelation at physiological conditions. Itgels in response to multiple in-situ environmental stimuli, and isreversible. It must have reasonable mechanical strength and havebiocompatibility with the host tissue.

The following references disclose processes or compounds useful in thisart:

-   U.S. Pat. No. 5,525,334-   U.S. Pat. No. 5,702,361-   U.S. Pat. No. 5,695,480-   U.S. Pat. No. 5,858,746-   U.S. Pat. No. 5,589,568-   T. G. Park and A. S. Hoffman, “Synthesis, Characterization, and    Application of pH/Temperature-sensitive Hydrogels”, Proceed. Intern.    Symp. Control. Rel. Bioact. Mater., 17 (1990), pp 112–113.-   G. Chen and A. S. Hoffman, “Graft Copolymers That Exhibit    Temperature-induced Phase Transitions Over a Wide Range of pH”, Vol    3, Nature, 1995, pp. 49–52.-   S. Beltran, J. P. Baker, H. H. Hooper, H. W. Blanch and J. M.    Prausnitz, “Swelling Equilibria for Weakly Ionizable,    Temperature-Sensitive Hydrogels”,Proc. Amer. Chem. Soc., 1991.-   J. Zhang and N. A. Peppas, “Synthesis and Characterization of pH-and    Temperature-Sensitive Poly(Methacrylic    acid)/Poly(N-isopropylacrylamide) Interpenetrating Polymeric    Networks, Macromolecules, 2000 (currently available on-line on the    world wide web).-   T. G. Park, ”Temperature Modulated Protein Release From    pH/Temperature Sensitive Hydrogels; Biomaterials 20 (1999), pp.    517–521.

SUMMARY

The present invention comprises an aqueous polymeric solution capable ofgelling upon exposure to a critical minimum value of a plurality ofenvironmental stimuli. A “plurality” of environmental stimuli may be anynumber equal to or greater than two, although in most cases the processof the present invention will utilize two stimuli. The aqueous polymericsolution is capable of having gelation reversed if all, or at a minimum,at least one, of the environmental stimuli fall below (or outside arange of) critical minimum values. The environmental stimuli may be anystimulus that induces gelation, and is exemplified by temperature, pH,ionic strength, electrical field, magnetic field, solvent composition,chemical composition, light, pressure and the like. The critical minimumvalues for each of these stimuli may vary depending upon the localenvironment in which the gel is used, and will likely be markedlydifferent between medical (or, in vivo) and industrial uses.

In vivo environmental stimuli may be either associated with humanpatients, or in veterinarian use with domestic or farm animals. Forexample, the product and process of the present invention may be usedwith cattle, horses, sheep, pigs, dogs, cats, and the like. Theenvironmental stimuli may be either conditions that are naturally foundwithin the area of use (e.g. “ambient”), or they may be externallyimposed. Generally speaking, when injected into a human, the aqueouspolymeric solution of the present invention is injected into a specificlocus within the body—either a cavity (such as a post-operative tumorsite) or a conduit/duct (such as a blood vessel) or into a tissue mass(such as a tumor).

The polymeric solution may either be a carrier for a pharmaceuticallyactive therapeutic agent (in which case it will typically be an aqueoussolution), or it may merely act as an inert blocking mass. An example ofthe former is the injection of chemicals or radioisotopes deliveredthrough a catheter to a tumor mass; an example of the latter isinjection of a gelled mass into a tubular body so as to cause arestriction therein (e.g. an aneurism of a blood vessel or the vasdeferens for purposes of reversible sterilization in males). Likewise,the polymeric solution may be used in industrial situations wherein itmay not be an aqueous solution.

It would be of great medical benefit in in vivo environments, if anaqueous polymeric solution could be transported in a catheter within thebody for extended distances without gelation. For example, if it isdesired to implant a quantity of radioisotope in a gelled mass withinthe brain, a catheter may be inserted into the patients femoral arteryand the therapeutic agent is transported from that locale to the brain.Through use of the process of the present invention, for example, thetwo stimuli to induce gelation may be temperature and pH in the bloodstream, such that warming of the liquid polymeric compound alone (withinthe catheter) will not cause gelation. It is not until the compoundcontacts the blood in the brain, and is induced by the pH or ionicstrength of the blood to gel, that gelation occurs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of the logarithm of storageviscosity (log n′) versus temperature for poly(NIPAAm-co-DMAEA) polymer;

FIG. 2 is a graphical representation of the storage modulus (G′) versustemperature for poly(NIPAAm-co-DMAEA) polymer;

FIG. 3 is a graphical representation of the complex viscosity logarithm(log n*) versus temperature for poly(NIPAAm-co-DMAEA) polymer;

FIG. 4 is a graphical representation of the storage viscosity logarithm(log n′) versus temperature for poly(NIPAAm-co-AAc) polymer;

FIG. 5 is a graphical representation of the storage modulus (G′) versustemperature for poly(NIPAAm-co-Aac) polymer; and

FIG. 6 is a graphical representation of the complex viscosity logarithm(log n*) versus temperature for poly(NIPAAm-co-Aac) polymer.

DETAILED DESCRIPTION

The reversible, stimulus-sensitive gels of the present invention areintended primarily for use in medical environments, such as theembolization of blood vessels at remote anatomical locations. However,it is to be understood that the gels of the present invention are notlimited to medical applications—they will find uses outside medicine.For example, uses as drilling muds in the drilling of oil or other deepwells; subterranean use to block transport of noxious pollutants in anaquifer; the sealing of any industrial conduit to block passage ofmaterials therein, and the like.

There are specific uses of gelling polymers wherein it is greatlypreferred that the gelation response occur only when the polymersolution is exposed to multiple stimuli. As used herein, the stimuli maybe any environmental stimulus that, in combination with at least oneadditional stimulus, causes the gelation reaction of a polymer solution.For example, such stimuli as temperature, pH, ionic strength, electricalfield, magnetic field, solvent concentration/composition, surroundingchemical concentration/composition, light or pressure. While it isprobably preferred in most cases that gelation result from two stimuli,it is within the context of the present invention that gelation occur inresponse to three or more stimuli.

As used herein, the stimuli responsible for gelation must reach a“critical minimum value” to effectively cause gelation. Values outsideof this critical minimum value will cause the polymer composition toflow as (or similar to) a liquid. The critical minimum value will dependupon the particular environment—and the value for a single stimuli (forexample, temperature) may be radically different depending upon the use.For example, in the context of a polymer solution injected into theblood stream of a human body wherein temperature and pH are the stimuliresponsible for gelation, the critical minimum value for temperature isapproximately 35–37° C. (internal body temperature) and the criticalminimum value for pH is approximately 7.0–7.5 (the pH of blood and manyother interstitial fluids). On the other hand, the critical minimumvalue for gelation in oil-field applications may be in excess of 70° C.and pH of less than 6.0 or greater than 8.0. For ease of description,and as described herein, any stimuli condition that falls outside thatrequired for gelation is denoted as “below” or “less than” the criticalminimum value, even though the value may be higher. For example, whileroom temperature may be on the order of 20° C. and therefore below thecritical minimum value (37° C.) to induce gelation, a temperature of 50°C. may likewise inhibit gelation because it is too high, and as definedherein is also “below” the critical minimum value.

Gelation is the change in viscosity from a fluid-like composition to asolid-like composition. While the degree of “solidness” may vary fromapplication to application, generally speaking gels of the presentinvention will exhibit viscosities in the full range of from paste-liketo solid-like.

In certain situations, it is critical that gelation of the gel bereversible. For example, the pre-operative embolization of vessels fortumor treatment may be necessary to successfully shrink such tumors; itis not desired that blood flow be forever blocked in such vessels due tosevere tissue damage. Upon return to environmental stimuli conditionsthat are “below” the critical minimum values, the gel reverses itsviscosity and returns to a solution that is transportable within itsimmediate environment. The gels of the present invention are highlystable and do not exhibit phase separation upon standing or uponrepeated cycling between the liquid and gel state. It is especiallyimportant that once gelled in situ, that the gel composition remaingelled indefinitely, or until intentionally reversed. For example, it isanticipated that gels of the present invention can be designed that willremain gelled for as long as many years.

Also, as used herein, the word “environmental” refers to the myriad ofstimuli that might induce gelation. In industrial, non-medical settingssuch environmental stimuli may comprise chemical composition,temperature, light, pressure, and the like. In the context of human orother mammalian bodies, the word “environmental” typically refers towell-known conditions within the body that can impact the gel(temperature, pH, ionic strength, etc.).

The polymers useful in the present invention include but are not limitedto thermally reversible copolymers that are useful as a gel that formswithout substantial syneresis when the thermally reversible copolymer isin an aqueous solution. Syneresis is defined as water expelled from acopolymer matrix upon gelation. Substantial syneresis is more than about10 wt % water expelled from the copolymer matrix. According to thepresent invention, it is preferred that the syneresis be less than about10 wt %, more preferably less than about 5 wt % and most preferably lessthan about 2 wt %. Substantially no syneresis is syneresis of less thanabout 2 wt %, preferably 0 wt %.

As an example of the sort of polymers that can be synthesized accordingto the present invention, and not intending to be limited by therecitation of specific compounds, the thermally reversible copolymer canbe a linear random, block or graft copolymers of an [meth-]acrylamidederivative and a hydrophilic comonomer wherein the copolymer is in theform of a plurality of chains having a plurality of molecular weightsgreater than or equal to a minimum geling molecular weight cutoff.According to the present invention, the minimum geling molecular weightcutoff is at least several thousand and is preferably about 12,000. Thepresence of a substantial amount of copolymer or polymer chains havingmolecular weights less than the minimum geling molecular weight cutoffresults in a milky solution that does not gel. Further, the amount ofhydrophilic comonomer in the linear random copolymer is preferably lessthan about 10 Mole %, more preferably less than about 5 Mole % and mostpreferably about 2 Mole %. When the hydrophyllic comonomer is AAc andthe thermosensitive co-monomer is NiPAAm, the amount of AAc in thelinear random copolymer is preferably from about 1 mole % to about 2.5Mole %, most preferably from about 1.6 Mole % to about 1.9 Mole %. Thestructure of linear chains is not cross linked. Moreover, the block orgraft copolymer structure is one in which a linear chain is shared byrandomly alternating portions of the [meth-]acrylamide derivative andthe hydrophilic comonomer.

The [meth-]acrylamide derivative is an N-alkyl substituted[meth-]acrylamide including but not limited toN-isopropyl[meth-]acrylamide, N,N-diethyl[meth-]acrylamide,N-[meth-]acryloylpyrrolidine, N-ethyl[meth-]acrylamide, and combinationsthereof.

The hydrophilic comonomer is any hydrophilic comonomer thatco-polymerizes with the [meth-]acrylamide derivative. Preferredhydrophilic comonomers are hydrophilic [meth-]acryl-compounds includingbut not limited to carboxylic acids, [meth-]acrylamide, hydrophilic[meth-]acrylamide derivatives, hydrophilic [meth-]acrylic acid esters.The carboxylic acid may be, for example, acrylic acid, dimer of acrylicacid, methacrylic acid and combinations thereof. The hydrophilicacrylamide derivatives include but are not limited toN,N-diethyl[meth-]acrylamide,2-[N,N-dimethylamino]ethyl[meth-]acrylamide,2-[N,N-diethylamino]ethyl[meth-]acrylamide, or combinations thereof. Thehydrophilic [meth-]acrylic esters include but are not limited to2-[N,N-diethylamino]ethyl [meth-]acrylate,2-[N,N-dimethylamino]ethyl[meth-]acrylate, and combinations thereof.

The polymer composition most likely having primary application inmedical applications is a hydrogel, wherein water is the solvent.Obviously, introducing non-aqueous solvents into a human or othermammalian body can have significant side effects. However, in industrialsettings, the solvent may comprise any well-known organic solvent.

In medical applications, the gels of the present invention may beutilized to deliver therapeutic agents to various body locations,including but not limited to intravenous and subcutaneous therapies,tissue supplementation, parenteral delivery, vascular and therapeuticembolization, tumor therapy, blockage of bodily conduits, and the like.Any biologically active compound having therapeutic qualities may bedelivered by the process of the present invention, including but notlimited to proteins, polypeptides, polynucleotides, polysaccharides,glycoproteins, lipoproteins, and the like.

Classes of therapeutically active or diagnostic compounds that will mostlikely be administered by the process of the present invention includebut are not limited to anti-cancer drugs, radionuclides, antibiotics,immunosuppressants, neurotoxins, antiinflammatory agents, imagingagents, and the like.

It is to be understood that while the biological uses of the productsand processes of the present invention will find particular applicationwith humans, other types of animals may be similarly treated. Becausethe cost of these procedures is relatively expensive, they typicallywill not be useful in a commercial sense with a broad range of animals.However, research applications of this technology with non-mammaliansmay be feasible. Other than humans, the invention will find particularapplication with cattle, horses, sheep, pigs, dogs, cats, and the like.

Generally speaking, compositions of polymers of the present inventionwill be found in very broad ranges. A reversible geling solution may bemade by mixing the reversible polymer with an aqueous solution in anamount of about 70 wt % to 99 wt %.

EXAMPLE 1

N-isopropylacrylamide was recrystallized from n-hexane and dried undervacuum. Acrylic acid was distilled under reduced pressure.2,2′-azobisisobutyronitrile was purified by recrystallization frommethanol. Dioxane was sonicated, degased and purged with deoxygenatednitrogen prior to use. Either nad hexane (reagent grade) were used asreceived. Phosphate-buffered saline (PBS) (pH=7.4) was made bydissolving 0.272 g of anhydrous KH₂PO₄, 2.130 g of Na₂HPO₄xH₂O and 8.474g of NaCl in 1.0 liter of ultra-pure water. pH of the solution wasadjusted to 7.4 with ORION 720A pH-meter.

The copolymers were obtained by free-radical solution copolymerizationof N-isopropylacrylamide (NIPAAm) with a proper comonomer;2-(dimethylamino)ethyl acrylate (DMAEA) for KK-11copolymer and acrylicacid (Aac) for Mj-114 copolymer. A positively ionizable, weakly basiccopolymer was synthesized in dioxane, using 97/3 mol % ratio of NIPAAmand DMAEA, using AIBN as a free-radical initiation. The monomers (5.000g, 4.415×10⁻² moles of NIPAAm and 207.3uL, 1.365×10⁻³ moles of DMAEA)were dissolved in dry, degassed dioxane (24 mL) and flushed with dry,deoxygenated nitrogen for 0.5 hour. After adding AIBN (4.8 mg, 2.93×10⁻⁵moles in dioxane solution (about 100 uL), the mixture was purged withnitrogen for additional 10 minutes. The polymerization was conducted at70° C. for 19 hr under pure nitrogen. The reaction mixture was thencooled to RT, diluted with dioxane (24 mL), poured into 3/1 v/v mixtureof ethyl ether/hexane and vigorously stirred for about 2 hours. Thecrude polymer was then filtered, washed with ether and dried in vacumovernight. Dry polymer was dissolved in 200 mL of UP water and filteredthrough a nylon membrane (pore size 0.45 um). Crude polymer solution waspurified by ultrafiltration (three times) using a 30KD MWCO membrane.The purified solution was freeze-dried to obtain a dry polymer powder(yield 84–85%).

The molar masses were analyzed by Gel Permeation Chromatography (GPC),using the following equipment:

-   -   two styragel columns, HMW 6E and HR 4E (7.8×300 mm both);    -   Rheodyne 50 or 200 ul loop injector;    -   Detectors: miniDAWN light scattering detector and Waters 410        Differential Refractometer;    -   515 HPLC Waters pump and isocratic THF (HPLC grade) mobile        phase, sonicated and degassed; flow rate was set at 0.5 ml/min;    -   Astra 4.70 software.        The results are summarized in Table 1 below.

TABLE 1 Molar mass analysis by gel permeation chromatography SampleAverage Molar Poydispersity ID Polymer ID mass (M_(w)), g/mol(M_(w)/M_(n)) MJ-114 poly(NIPA-co-AAc) 1.14 ± 0.03 e+05 1.024 ± 0.04KK-11 poly(NIPA-co- DMAEA) 6.37 ± 0.48 e+05  1.05 ± 0.08

Reversible sol-gel transitions of the poly(NIPAAm-co-DMAEA) andpoly(NIPAAm-co-Aac) polymer solutions were studied using dynamicrheology (Rheometric Scientific SR 2000). The polymer solutions wereplaced between parallel plates with diameter 2.5 mm and gap 0.5 mm. TheDynamic Temperature Ramp Test (DTRT) was conducted under controlledstress (2.0 dync/cm²) and frequency (1.0 radian/sec.). Theheating-cooling cycle temperature gap was established for 21–37° C. withincrement 0.3° C. A 10% polymer solution in water and PBS wasinvestigated.

FIGS. 1–6 illustrate the results of DTRT conducted forpoly(NIPAAm-co-DMAEA) and poly(NIPAAm-co-AAc) polymer solutions attemperature gap 21–37° C. FIG. 1 illustrates changes in the log of thestorage viscosity (n′) of poly(NIPAAm-co-DMAEA) (KK-11) polymer solutionas a function of temperature. Heating process (H) causes sol to geltransition and, as a result, viscosity increases by three orders ofmagnitude (0.1–100 Pa×s). The increase is very sharp and takes place atabout 34–36.5° C. Upon cooling (C), the gel melts at a temperature thatis lower than the gelation temperature indicating characteristichysteresis loop between gel formation and gel melting temperatures. Thisbehavior results from resistance to disintegration of entangled hydrogelmolecules. This experiment was conducted under controlled stress (2.0dyne/cm²) and frequency (1.0 radian/sec). The heating-cooling cycletemperature increment was 0.3° C. FIG. 2 illustrates how the storagemodulus changes as a function of temperature, under controlled stress(2.0 dyne/cm²) and frequency (1.0 radian/sec). The heating-cooling cycletemperature increment was 0.3° C. FIG. 3 illustrates the logarithm ofcomplex viscosity changes as a function of temperature, conducted undercontrolled stress (2.0 dyne/cm²) and frequency (1.0 radian/sec). Theheating-cooling cycle temperature increment was 0.3° C.

FIGS. 4–6 illustrate rheological behavior of poly(NIPAAm-co-Aac) (MJ114k) polymer solution. Each of the experiments of FIGS. 4–6 wereconducted under controlled stress (2.0 dyne/cm 2) and frequency (1.0radian/sec.). The heating-cooling cycle temperature increment was 0.3°C.

The properties illustrated in FIGS. 1–6 illustrate the benefits of theinstant invention. A sharp sol to gel transition takes place just beforethe physiological temperature of the human body, and hysteresis helps toavoid quick melting of the formed gel due to small fluctuations of bodytemperature. The storage modulus (G′) of two gels is practically zero ata sol state, so it is not shown on a heating curve. It appears andsharply increases at 32.0 and 32.5° C. for poly(NIPAAm-co-Aac) andpoly(NIPAAm-co-DMAEA) gels solutions in PBS, as shown in FIGS. 2 and 5respectively. For both gels, the maximum value of the storage modulus isat about 37° C., indicating that the material is susceptible forinjectable gelling formulations.

Different behavior of the polymer solutions in water versus those in PBSproves that it is possible to deliver the water solution to a remoteanatomical location via a long needle or catheter in a sol (non-gelled)state. The polymer water solution will gel upon contact with body fluidsat physiological temperature.

EXAMPLE 2

A copolymer of N-isopropylacrylamide with hydrophilic comonomer,2-(dimethylamino)ethyl acrylate (DMAEA) was synthesized in dioxane by afree radical polymerization. After a two-step extensive purification, byprecipitation and ultrafiltration the NDAEA copolymer was lyophilized toobtain the copolymer in a powder form. This powder was then dissolved inwater to form an aqueous solution.

A 2 ml sample of this solution, warmed up to 37° C., was placed in a 2ml syringe equipped with a 30 Gauge needle. The needle was immersed in aphosphate buffered saline (PBL) solution also warmed up to 37° C. Thewarm copolymer solution from the syringe was injected into the warm PBSsolution. Instantaneous gel formation was observed; the injected gelformed a “string”.

An additional 2 ml sample of the same aqueous solution was heatedgradually from room temperature up to 40° C. No gel formation wasobserved even at 40° C.

Yet another 2 ml sample of the same solution, equilibrated at roomtemperature, was placed in a 2 ml syringe equipped with a 30 Gaugeneedle. The needle was immersed in a phosphate buffered saline (PBs)solution also equilibrated at room temperature. The room temperaturecopolymer solution from the syringe was injected into the roomtemperature PBS solution. No gel formation was observed—the injectedpolymer solution simply dissolved in the buffer.

Thus, the impact of multiple stimulus gelation is evident in temperatureand proper ionic strength is required to cause gelation of the NDAEAcopolymer. Change in only one stimuli was ineffective to cause gelation.

EXAMPLE 3

A copolymer of N-isopropylacrylamide with hydrophilic comonomer,2-(N,N-dimethylamino)ethyl acrylate (NDAEA) was synthesized in dioxaneby a free-radical polymerization. After a two-step extensivepurification, by precipitation and ultrafiltration, the NDAEA copolymerwas lyophilized to obtain the copolymer in powder form. This powder wasthen dissolved in water to form an aqueous solution.

A 2 ml sample of this solution, warmed to 37° C., was placed in a 2 mlsyringe equipped with a 30 Gauge needle. The needle was immersed in aPBS solution also warmed up to 37° C. The warm copolymer solution fromthe syringe was injected into the warm PBS solution. Instantaneous gelformation was observed, the injected gel forming a “string”. Another 2ml sample of the same aqueous solution was heated gradually from roomtemperature up to 40° C. No gel formation was observed even at 40° C.

Yet another 2 ml sample of the solution, equilibrated at roomtemperature, was placed in a 2 ml syringe equipped with a 30 Gaugeneedle. The needle was immersed in a PBS solution also equilibrated atroom temperature. The room temperature copolymer solution from thesyringe was injected into the room temperature PBS solution. No gelformation was observed; the injected polymer solution simply dissolvedin the buffer.

CLOSURE

Having thus described a preferred exemplary embodiment of the presentinvention, it should be noted by those skilled in the art that thedisclosures herein are exemplary only and that alternatives, adaptationsand modifications may be made within the scope of the present invention.Thus, the present invention is not to be limited to the specificembodiments illustrated herein, but solely by the scope of the claimsappended hereto.

1. A method of reversibly sterilizing a mammal, comprising providing apolymeric compound comprising a methacrylamide derivative and ahydrophilic comonomer, that remains a solution until exposed to criticalminimum values of at least two environmental stimuli, wherein thepolymeric compound forms a gel upon exposure to the critical minimumvalues of the at least two environmental stimuli; delivering thepolymeric compound to a lumen or other body region in need of closurewhen the polymeric compound is a solution; and exposing the polymericcompound to the critical minimum values of the at least twoenvironmental stimuli such that the polymeric compound forms a gel insitu in the lumen or other body region resulting in reversiblesterilization of the mammal.
 2. The method of claim 1, wherein thesterilization of the mammal is reversed when one of the at least twoenvironmental stimuli falls below the critical minimum value.
 3. Themethod of claim 1, wherein the exposing the polymeric compound to thecritical minimum values of the at least two environmental stimulicomprises exposing the polymeric compound to at least two environmentalstimuli selected from the group consisting of temperature, pH, ionicstrength, electrical field, magnetic filed, solvent composition, light,pressure and chemical composition of the ambient environment.
 4. Amethod for sterilizing a male mammal wherein the sterilization isreversible, comprising: delivering a polymeric compound comprising amethacrylamide derivative and a hydrophilic comonomer, that remains asolution until exposed to critical minimum values of at least twoenvironmental stimuli, and forms a gel upon exposure to the criticalminimum values of the at least two environmental stimuli, to vasdeferens of the male mammal when the polymeric compound is a solution;and exposing the polymeric compound to the critical minimum values ofthe at least two environmental stimuli such that the polymeric compoundforms a gel in situ in the vas deferens thereby reversibly sterilizingthe male mammal.
 5. The method of claim 4, wherein the sterilization ofthe mammal is reversed when one of the at least two environmentalstimuli falls below the critical minimum value.